Detection of variations in human H2 receptors

ABSTRACT

A group of oligonucleotides for the identification of sequences in a sample comprising human histamine H 2  receptor DNA, cDNA or RNA originating from a tissue sample or body fluid is employed in the diagnosis and/or treatment of human neurological and psychiatric disorders, particularly schizophrenia, and diseases of other systems or organs of the human body. The oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H 2  receptor, have nucleotide sequences selected from: 5′ACACCAGCCTGGATGTGA 3′(as listed in SEQ ID NO:12), 5′TCACATCCAGGCTGGTCT 3′ (as listed in SEQ ID NO:13), 5′ CAATCATACCACCTCTAA 3′ (as listed in SEQ ID NO:14), 5′ ACACAAACGCGGTGAAGT 3′(as listed in SEQ ID NO:15). Also described is a diagnostic kit comprising one or more of the above mentioned oligonucleotides.

This is a International Patent Application No. PCT/GB97/01075, with an international filing date of Apr. 18, 1997, now pending.

TECHNICAL FIELD

This invention relates to the detection of variations in human histamine H₂ receptors, and more particularly to the development of new compounds useful in the sequencing and identification of a human histamine H₂ receptor and their use in the diagnosis and treatment of certain human disorders, for example, brain disorders. The invention also relates to new compounds and a method for detecting an allelic polymorphic variation within the human population for the gene encoding the histamine H₂ receptor, and their use in the diagnosis and treatment of human disorders.

BACKGROUND OF THE INVENTION

The histamine H₂ receptor is one of many receptors in the body. Compounds used to treat many diseases work by activating a receptor or inhibiting the action of its natural ligand. Variations in some receptors amongst the population are known to be caused by allelic variation, and this variation can alter the response of a disease to a drug amongst patients. An example of this would be the response to clozapine, used to treat schizophrenia, associated with allelic variation in the 5-HT_(2A) receptor demonstrated by Arranz el al (1995) Lancet, 346(8970), 281-282.

In co-pending UK Patent Applications Nos. GB9503866.7 and GB9522889.6, and in International Patent Application No. PCT/EP96/00397, it is reported that there is a hitherto unrecognised allele or subtype of the human histamine H₂ receptor gene which may be specific to the brain.

In the above mentioned co-pending patent specifications there are described and claimed:

1) a sequence for a novel allele of a human histamine H₂ receptor gene comprising up to six single base substitutions compared with the cDNA sequence published by Gantz et al (1991) Biochem Biophys Res Comm 178,3,1386-1392 as follows:

site of change base change amino acid alteration 398 T-C Val-Gly 525 A-T Lys-Asn 620 A-G Lys-Asp 649 A-G Asn-Asp 692 A-G Lys-Arg 802 G-A Val-Met

The cDNA sequence in Gantz et al., supra, is:

   1 atggcaccca atggcacagc ctcttccttt tgcctggact ctaccgcatg caagatcacc (SEQ ID NO:18)   61 atcaccgtgg tccttgcggt cctcatcctc atcaccgttg ctggcaatgt ggtcgtctgt  121 ctggccgtgg gcttgaaccg ccggctccgc aacctgacca attgtttcat cgtgtccttg  181 gctatcactg acctgctcct cggcctcctg gtgctgccct tctctgccat ctaccagctg  241 tcctgcaagt ggagctttgg caaggtcttc tgcaatatct acaccagcct ggatgtgatg  301 ctctgcacag cctccattct taacctcttc atgatcagcc tcgaccggta ctgcgctgtc  361 atggacccac tgcggtaccc tgtgctggtc accccagttc gggtcgccat ctctctggtc  421 ttaatttggg tcatctccat taccctgtcc tttctgtcta tccacctggg gtggaacagc  481 aggaacgaga ccagcaaggg caatcatacc acctctaagt gcaaagtcca ggtcaatgaa  541 gtgtacgggc tggtggatgg gctggtcacc ttctacctcc cgctactgat catgtgcatc  601 acctactacc gcatcttcaa ggtcgcccgg gatcaggcca agaggatcaa tcacattagc  661 tcctggaagg cagccaccat cagggagcac aaagccacag tgacactggc cgccgtcatg  721 ggggccttca tcatctgctg gtttccctac ttcaccgcgt ttgtgtaccg tgggctgaga  781 ggggatgatg ccatcaatga ggtgttagaa gccatcgttc tgtggctggg ctatgccaac  841 tcagccctga accccatcct gtatgctgcg ctgaacagag acttccgcac cgggtaccaa  901 cagctcttct gctgcaggct ggccaaccgc aactcccaca aaacttctct gaggtccaac  961 gcctctcagc tgtccaggac ccaaagccga gaacccaggc aacaggaaga gaaacccctg 1021 aagctccagg tgtggagtgg gacagaagtc acggcccccc agggagccac agacaggtaa 1081 aagctccagg tgtggagtgg gacagaagtc acggcccccc agggagccac agacaggtaa 1141 gcgctgaaca gagacttccg caccgggtac caacagctct tctgctgcag g

This cDNA sequence encodes the following amino acid sequence:

MAPNGTASSFCLDSTACKITITVVLAVLILITVAGNVVVCLAVGLNRRLRNLTNCFIVSLAITDLLLGLLVLPFSAI (SEQ ID NO: 19) YQLSCKWSFGKVFCNIYTSLDVMLCTASILNLFMISLDRYCAVMDPLRYPVLVTPVRVAISLVLIWVISITLSFLSI HLGWNSRNETSKGNHTTSKCKVQVNEVYGLVDGLVTFYLPLLIMCITYYRIFKVARDQAKRINHISSWKAATIREHK ATVTLAAVMGAFIICWFPYFTAFVYRGLRGDDAINEVKEAIVKWLGYANSALNPILYAAKNRDFRTGYQQLFCCRLA NRNSHKTSLRSNASQLSRTQSREPRQQEEKPLKLQVWSGTEVTAPQGATDR

2) a nucleotide sequence coding for a region of a human histamine H₂ receptor, comprising one or more of the following base substitutions compared with the published sequence in Gantz et al (1991) supra, and from which the positional notation is taken:

site of change base 398 C 525 T 620 G 649 G 692 G 802 A

The nucleotide sequence can, for example, comprise the following sequence (as listed in SEQ ID NO: 1):

5′  CAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTGTC     CTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCAAT     CATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGATG     GGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGCAT     CTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGAAG     GCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGGGG     CCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTGAG     AGGGGATGATGCCATCAATGAGATGTTA  3′

As a specific exemplification, the nucleotide sequence can comprise the following sequence (as listed in SEQ ID NO: 2):

5′  CCAATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCAT     CACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTC     TGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCG     TGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGC     CATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTAC     ACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCA     GCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCAC     CCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTG     TCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCA     ATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGA     TGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGC     ATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGA     AGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGG     GGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTG     AGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCT     ATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCG     CACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAA     ACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCA     GGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG  3′

3) new oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H₂ receptor, having nucleotide sequences selected from:

1) 5′ CCAATGGCACAGCCTCTT 3′ (as listed in SEQ ID NO: 3)

2) 5′ CGTGACTTCTGTCCCACT 3′ (as listed in SEQ ID NO: 4)

3) 5′ CCAGGCAACAGGAAGAGA 3′ (as listed in SEQ ID NO: 5)

4) 5′ TCTCTTCCTGTTGCCTGG 3′ (as listed in SEQ ID NO: 6)

5) 5′ GCAGCAGAAGAGCTGTTG 3′ (as listed in SEQ ID NO: 7)

6) 5′ TCCAGGTCAATGAAGTGT 3′ (as listed in SEQ ID NO: 8)

7) 5′ ACACTTCATTGACCTGGA 3′ (as listed in SEQ ID NO: 9)

8) 5° CCAAGAGGATCAATCACA 3′ (as listed in SEQ ID NO: 10)

9) 5′ TGTGATTGATCCTCTTGG 3′ (as listed in SEQ ID NO: 11)

and

4) a diagnostic kit comprising one or more of the above new oligonucleotides.

DISCLOSURE OF THE INVENTION

The present invention is concerned in one aspect with improvements in the diagnosis and/or treatment of human neurological and psychiatric disorders, and more particularly in the diagnosis and treatment of schizophrenia. In another aspect, the invention is concerned with improvements in the diagnosis and/or treatment of diseases of other systems or organs of the human body.

According to the invention, a further group of oligonucleotides has now been developed for the identification of sequences in a sample comprising a human histamine H₂ receptor DNA, cDNA or RNA originating from a tissue sample or body fluid.

In a first aspect, therefore, the invention provides new oligonucleotides, suitable for use as primers for the amplification of DNA corresponding to a region of a human histamine H₂ receptor, having nucleotide sequences selected from:

10) 5′ ACACCAGCCTGGATGTGA 3′ (as listed in SEQ ID NO:12)

11) 5′ TCACATCCAGGCTGGTGT 3′ (as listed in SEQ ID NO:13)

12) 5′ CAATCATACCACCTCTAA 3′ (as listed in SEQ ID NO:14)

13) 5′ ACACAAACGCGGTGAAGT 3′ (as listed in SEQ ID NO:15)

15 and a diagnostic kit comprising one or more of the above mentioned oligonucleotides.

The direction and base start numbers for the novel oligonucleotide primers are as follows:

Primer Base Start No. 10) Upstream 281 11) Downstream 298 12) Upstream 501 13) Downstream 766

Information on the human histamine H₂ receptor was obtained from the MRC Daresbury database accessing “Genem 61” File no. M64799—Human histamine H₂ receptor gene.

The above mentioned substitutions in the human histamine H₂ receptor gene alter and in some instances introduce or remove new sites for cleavage by specific restriction endonucleases as follows:

base change site alters restriction map of: 398 AluI, AvaI, BspWI, BsrI, CviJI 525 620 Eco57 649 ClaI, Sau3A, TaqI 692 802 MnlI

In another aspect the invention provides a diagnostic kit comprising one or more of the new oligonucleotide primers and, preferably, one or more of the above mentioned endonucleases, optionally with one or more buffers.

In a still further aspect, the invention provides a diagnostic kit comprising one or more of the new oligonucleotide primers whereby detection of human histamine subtypes can be achieved using single-stranded conformational polymorphism (SSCP). The kit can, for example, comprise one or more of the above mentioned oligonucleotide primers, and, optionally, formamide.

A kit may be used to establish genotype or base variations. This information may be used in predicting an individual's disease susceptibility, disease course, prognosis and/or response to treatment, as would be understood by those skilled in the art from the disclosure contained herein. The treatment response or efficacy which may be predicted may include drug treatment such as for example, use of H₂ receptor antagonists, for example, famotidine, or other forms of treatment such as social or psychological intervention.

Eucaryotic expression vectors and host cells transformed thereby can be prepared as described in the above mentioned co-pending patent specifications and the encoded protein analysed.

The invention is illustrated by the following Examples:

EXAMPLE 1

This example describes the identification and sequencing of an allelic human H₂ receptor gene using certain novel oligonucleotide primers according to the invention.

A polymerase chain reaction (PCR) product is prepared from human DNA.

DNA was extracted from human brain tissue by first pulverizing approximately 1g of tissue in liquid nitrogen then adding to 10 ml lysis buffer (0.32M sucrose, 10 mM Tris, 5 mM magnesium chloride 1% Triton X-100 pH8.0). This solution was centrifuged (9,000 rpm 15 mins) to pellet the tissue, the lysis buffer was drawn off and the pellet resuspended in 4.5 ml 75 mM sodium chloride, 24 mM EDTA. This solution was then incubated for 3 hours with 250 μl 10% SDS and 2 mg proteinase K at 56° C. This aqueous phase was then extracted twice with 5 ml of phenol:chloroform:isoamyl alcohol (25:24:1). Then sodium acetate to 0.3M pH7.5 and 2 volumes of ethanol (at −20° C.) were added to the aqueous phase and the DNA hooked out into TE buffer. The concentration of the DNA was determined by measuring the optical density of the sample, at a wavelength of 260 nm.

The DNA was then amplified by the polymerase chain reaction using the oligonucleotide primers 1) and 2) (as hereinbefore described) for 36 cycles. The timing for each cycle was as follows; 1 min at 94° C., 1.5 min at 56° C. and 2 mins at 72°C., this was then followed by a 10 min extension at 72° C. (Amplitaq DNA polymerase Perkin-Elmer Cetus). This reaction produced a DNA fragment of 1047 base pair when analyzed by gel electrophoresis.

Following PCR amplification of the DNA, the PCR products were immediately ligated and cloned into the TA cloning system (InvitroGen). The transformed cells were plated onto Luria-Bertani plates containing 50 μl/ml amplicillin and 1.6 mg X-Gal. Plates were then incubated overnight at 37° C., then moved to 4° C. for 4 hours to allow for colour development. Positive (white colonies) were then analyzed by growing a 5 ml culture overnight at 37° C. extracting the plasmids (Qiaspin minipreps (Qiagen)) and performing an EcoRI digest to ensure the correct size product was contained in the plasmid. The plasmid used to clone the PCR product is the PCR™ II Vector, which is transformed into One Shot™ INVαF′ Competent cells.

Both strands of the cloned PCR product were sequenced using the dideoxynucleotide chain-terminated method, carried out with Sequenase version 2.0 (Amersham/USB). Partial sequencing of short stretches of the cloned DNA utilised all the oligonucleotide primers 1) to 11) hereinbefore described. The cloned PCR product was shown to be identical to the gastric cDNA of Gantz et, al except for the previously mentioned six single base changes.

Results and Discussions

The sequence derived from the above described method is listed below and in SEQ ID NO: 2. The novel form of the human Histamine H₂ receptor gene is designated the H₂R_(549G) allele (SEQ ID NO:2), and the original form as described by Gantz et al is designated the H₂R_(649A) allele, as described by Orange et al (1996) Neuro Report 7(7) (to be published).

5′  AATGGCACAGCCTCTTCCTTTTGCCTGGACTCTACCGCATGCAAGATCACCAT     CACCGTGGTCCTTGCGGTCCTCATCCTCATCACCGTTGCTGGCAATGTGGTCGTC     TGTCTGGCCGTGGGCTTGAACCGCCGGCTCCGCAACCTGACCAATTGTTTCATCG     TGTCCTTGGCTATCACTGACCTGCTCCTCGGCCTCCTGGTGCTGCCCTTCTCTGC     CATCTACCAGCTGTCCTGCAAGTGGAGCTTTGGCAAGGTCTTCTGCAATATCTAC     ACCAGCCTGGATGTGATGCTCTGCACAGCCTCCATTCTTAACCTCTTCATGATCA     GCCTCGACCGGTACTGCGCTGTCATGGACCCACTGCGGTACCCTGTGCTGGTCAC     CCCAGCTCGGGTCGCCATCTCTCTGGTCTTAATTTGGGTCATCTCCATTACCCTG     TCCTTTCTGTCTATCCACCTGGGGTGGAACAGCAGGAACGAGACCAGCAAGGGCA     ATCATACCACCTCTAAGTGCAATGTCCAGGTCAATGAAGTGTACGGGCTGGTGGA     TGGGCTGGTCACCTTCTACCTCCCGCTACTGATCATGTGCATCACCTACTACCGC     ATCTTCAGGGTCGCCCGGGATCAGGCCAAGAGGATCGATCACATTAGCTCCTGGA     AGGCAGCCACCATCAGGGAGCACAGAGCCACAGTGACACTGGCCGCCGTCATGGG     GGCCTTCATCATCTGCTGGTTTCCCTACTTCACCGCGTTTGTGTACCGTGGGCTG     AGAGGGGATGATGCCATCAATGAGATGTTAGAAGCCATCGTTCTGTGGCTGGGCT     ATGCCAACTCAGCCCTGAACCCCATCCTGTATGCTGCGCTGAACAGAGACTTCCG     CACCGGGTACCAACAGCTCTTCTGCTGCAGGCTGGCCAACCGCAACTCCCACAAA     ACTTCTCTGAGGTCCAACGCCTCTCAGCTGTCCAGGACCCAAAGCCGAGAACCCA     GGCAACAGGAAGAGAAACCCCTGAAGCTCCAGGTGTGGAGTGGGACAGAAGTCACG  3′

EXAMPLE 2

This example describes the identification and sequencing of an allelic human H₂ receptor gene using certain novel oligonucleotide primers according to the invention.

The example describes confirmation of the existence of polymorphisms in the human histamine H₂ receptor gene utilising single-stranded conformational polymorphism (SSCP). This is made possible by an assay based upon amplification of a 266 base pair PCR fragment of the human histamine H₂ receptor gene, followed by specific acrylamide gel electrophoresis. (it will be apparent to those skilled in the art that single base changes may also be detected by other techniques known to those in the art, including, chemical cleavage, PCR thermoligase reactions, restriction endonuclease digestion of human histamine H₂ receptor gene PCR fragments etc.)

Samples of blood are collected from human volunteers into EDTA coated tubes. 1 ml of this blood is heated to 100° C. for 15 minutes and then spun in a microcentrifuge at 13,000 Xg for 15 minutes. The supernatant is collected and the cell debris is discarded. 1 μl of the supernatant is utilised as template DNA for a PCR reaction to amplify a portion of the human histamine H₂ receptor gene between bases 501 and 766. The conditions for this PCR reaction are 3 mM MgCl₂(Gibco BRL), 1XPCR buffer (Gibco BRL), 0.2 mM of each dATP, dCTP, dTTP, dGTP (Gibco BRL) 10 pmoles of each of oligonucleotide primers 12) and 13) (hereinbefore described) and 1 unit Taq DNA polymerase, in a total volume adjusted to 50 μl by sterile DNAse free water. This mix is then subjected to the following conditions in a Progene thermal cycler (Techne); 96° C., 5 minutes, then 35 cycles of 96° C., 45 seconds, 56° C., 45 seconds, 72° C., 45 seconds.

10 μl of the resultant product is analysed on a 1% agarose gel to ensure that the above reaction is correctly amplifying the target DNA fragement. Then 7.5 μl of the PCR mix is added to 7.5 μl of 95% formamide, 10 mM EDTA pH 8.0, heated to 10020 C. for 5-10 minute, and then quenched immediately in slushy ice for 5 minutes. 3 μl of 6×blue/orange loading dye is then added (Promega) and the mix is loaded into a 1 mm thick 14% 59:1 (acrylamide:bis-acrylamide) electrophoresis gel containing 2% glycerol, prepared in 0.5×TBE buffer. The gel is run for 18-24 hours at a constant voltage of 260V, at room temperature. The gel is then fixed in 10% methanol, 10% ethanoic acid for 10 minutes. The gel is next placed into 1 mg/ml silver nitrate for 15 minutes, then rinsed briefly with distilled water. The gel is then placed into 0.26% formaldehyde, 0.38 M sodium hydroxide for 15 minutes. The gel is finally placed into 0.75% sodium carbonate for 10 minutes.

Typical results are illustrated diagrammatically in FIG. 1 which shows a SSCP gel, prepared as above described, on which different samples have been loaded. The following patterns of banding are seen:

Lane 1 shows a pattern corresponding to a homozygons H₂R_(649G) genotype.

Lane 2 shows patterns corresponding to an individual heterozygons H₂R_(649A)/H₂R_(649G) genotype.

Lane 3 shows a pattern corresponding to a homozygous H₂R_(649A) genotype.

Alternative embodiments of the invention can be envisaged by those skilled in the art from the Information contained herein. All such alternative embodiments are Intended to lie within the scope of this application.

The reader's attention is directed to all papers and documents which are filed concurrently with this specification and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps or any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

Each feature disclosed in this specification (including any accompanying claims, abstract and drawings), may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

The invention is not restricted to the details of the foregoing embodiments. This invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.

19 1 413 DNA Homo sapiens CDS (3)...(410) 1 ca gct cgg gtc gcc atc tct ctg gtc tta att tgg gtc atc tcc att 47 Ala Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile Ser Ile 1 5 10 15 acc ctg tcc ttt ctg tct atc cac ctg ggg tgg aac agc agg aac gag 95 Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser Arg Asn Glu 20 25 30 acc agc aag ggc aat cat acc acc tct aag tgc aat gtc cag gtc aat 143 Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gln Val Asn 35 40 45 gaa gtg tac ggg ctg gtg gat ggg ctg gtc acc ttc tac ctc ccg cta 191 Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro Leu 50 55 60 ctg atc atg tgc atc acc tac tac cgc atc ttc agg gtc gcc cgg gat 239 Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Arg Val Ala Arg Asp 65 70 75 cag gcc aag agg atc gat cac att agc tcc tgg aag gca gcc acc atc 287 Gln Ala Lys Arg Ile Asp His Ile Ser Ser Trp Lys Ala Ala Thr Ile 80 85 90 95 agg gag cac aga gcc aca gtg aca ctg gcc gcc gtc atg ggg gcc ttc 335 Arg Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala Phe 100 105 110 atc atc tgc tgg ttt ccc tac ttc acc gcg ttt gtg tac cgt ggg ctg 383 Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly Leu 115 120 125 aga ggg gat gat gcc atc aat gag atg tta 413 Arg Gly Asp Asp Ala Ile Asn Glu Met 130 135 2 1046 DNA Homo sapiens CDS (3)...(1046) 2 cc aat ggc aca gcc tct tcc ttt tgc ctg gac tct acc gca tgc aag 47 Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala Cys Lys 1 5 10 15 atc acc atc acc gtg gtc ctt gcg gtc ctc atc ctc atc acc gtt gct 95 Ile Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile Thr Val Ala 20 25 30 ggc aat gtg gtc gtc tgt ctg gcc gtg ggc ttg aac cgc cgg ctc cgc 143 Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg Leu Arg 35 40 45 aac ctg acc aat tgt ttc atc gtg tcc ttg gct atc act gac ctg ctc 191 Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp Leu Leu 50 55 60 ctc ggc ctc ctg gtg ctg ccc ttc tct gcc atc tac cag ctg tcc tgc 239 Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu Ser Cys 65 70 75 aag tgg agc ttt ggc aag gtc ttc tgc aat atc tac acc agc ctg gat 287 Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser Leu Asp 80 85 90 95 gtg atg ctc tgc aca gcc tcc att ctt aac ctc ttc atg atc agc ctc 335 Val Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile Ser Leu 100 105 110 gac cgg tac tgc gct gtc atg gac cca ctg cgg tac cct gtg ctg gtc 383 Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val Leu Val 115 120 125 acc cca gct cgg gtc gcc atc tct ctg gtc tta att tgg gtc atc tcc 431 Thr Pro Ala Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile Ser 130 135 140 att acc ctg tcc ttt ctg tct atc cac ctg ggg tgg aac agc agg aac 479 Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser Arg Asn 145 150 155 gag acc agc aag ggc aat cat acc acc tct aag tgc aat gtc cag gtc 527 Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gln Val 160 165 170 175 aat gaa gtg tac ggg ctg gtg gat ggg ctg gtc acc ttc tac ctc ccg 575 Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro 180 185 190 cta ctg atc atg tgc atc acc tac tac cgc atc ttc agg gtc gcc cgg 623 Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Arg Val Ala Arg 195 200 205 gat cag gcc aag agg atc gat cac att agc tcc tgg aag gca gcc acc 671 Asp Gln Ala Lys Arg Ile Asp His Ile Ser Ser Trp Lys Ala Ala Thr 210 215 220 atc agg gag cac aga gcc aca gtg aca ctg gcc gcc gtc atg ggg gcc 719 Ile Arg Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala 225 230 235 ttc atc atc tgc tgg ttt ccc tac ttc acc gcg ttt gtg tac cgt ggg 767 Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly 240 245 250 255 ctg aga ggg gat gat gcc atc aat gag atg tta gaa gcc atc gtt ctg 815 Leu Arg Gly Asp Asp Ala Ile Asn Glu Met Leu Glu Ala Ile Val Leu 260 265 270 tgg ctg ggc tat gcc aac tca gcc ctg aac ccc atc ctg tat gct gcg 863 Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr Ala Ala 275 280 285 ctg aac aga gac ttc cgc acc ggg tac caa cag ctc ttc tgc tgc agg 911 Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Leu Phe Cys Cys Arg 290 295 300 ctg gcc aac cgc aac tcc cac aaa act tct ctg agg tcc aac gcc tct 959 Leu Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn Ala Ser 305 310 315 cag ctg tcc agg acc caa agc cga gaa ccc agg caa cag gaa gag aaa 1007 Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gln Gln Glu Glu Lys 320 325 330 335 ccc ctg aag ctc cag gtg tgg agt ggg aca gaa gtc acg 1046 Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr 340 345 3 18 DNA Artificial Sequence Synthetically generated oligonucleotide 3 ccaatggcac agcctctt 18 4 18 DNA Artificial Sequence Synthetically generated oligonucleotide 4 cgtgacttct gtcccact 18 5 18 DNA Artificial Sequence Synthetically generated oligonucleotide 5 ccaggcaaca ggaagaga 18 6 18 DNA Artificial Sequence Synthetically generated oligonucleotide 6 tctcttcctg ttgcctgg 18 7 18 DNA Artificial Sequence Synthetically generated oligonucleotide 7 gcagcagaag agctgttg 18 8 18 DNA Artificial Sequence Synthetically generated oligonucleotide 8 tccaggtcaa tgaagtgt 18 9 18 DNA Artificial Sequence Synthetically generated oligonucleotide 9 acacttcatt gacctgga 18 10 18 DNA Artificial Sequence Synthetically generated oligonucleotide 10 ccaagaggat caatcaca 18 11 18 DNA Artificial Sequence Synthetically generated oligonucleotide 11 tgtgattgat cctcttgg 18 12 18 DNA Artificial Sequence Synthetically generated oligonucleotide 12 acaccagcct ggatgtga 18 13 18 DNA Artificial Sequence Synthetically generated oligonucleotide 13 tcacatccag gctggtgt 18 14 18 DNA Artificial Sequence Synthetically generated oligonucleotide 14 caatcatacc acctctaa 18 15 18 DNA Artificial Sequence Synthetically generated oligonucleotide 15 acacaaacgc ggtgaagt 18 16 136 PRT Homo sapiens 16 Ala Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile Ser Ile Thr 1 5 10 15 Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser Arg Asn Glu Thr 20 25 30 Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gln Val Asn Glu 35 40 45 Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro Leu Leu 50 55 60 Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Arg Val Ala Arg Asp Gln 65 70 75 80 Ala Lys Arg Ile Asp His Ile Ser Ser Trp Lys Ala Ala Thr Ile Arg 85 90 95 Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala Phe Ile 100 105 110 Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly Leu Arg 115 120 125 Gly Asp Asp Ala Ile Asn Glu Met 130 135 17 348 PRT Homo sapiens 17 Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala Cys Lys Ile 1 5 10 15 Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile Thr Val Ala Gly 20 25 30 Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg Leu Arg Asn 35 40 45 Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp Leu Leu Leu 50 55 60 Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu Ser Cys Lys 65 70 75 80 Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser Leu Asp Val 85 90 95 Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile Ser Leu Asp 100 105 110 Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val Leu Val Thr 115 120 125 Pro Ala Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val Ile Ser Ile 130 135 140 Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser Arg Asn Glu 145 150 155 160 Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Asn Val Gln Val Asn 165 170 175 Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr Leu Pro Leu 180 185 190 Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Arg Val Ala Arg Asp 195 200 205 Gln Ala Lys Arg Ile Asp His Ile Ser Ser Trp Lys Ala Ala Thr Ile 210 215 220 Arg Glu His Arg Ala Thr Val Thr Leu Ala Ala Val Met Gly Ala Phe 225 230 235 240 Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr Arg Gly Leu 245 250 255 Arg Gly Asp Asp Ala Ile Asn Glu Met Leu Glu Ala Ile Val Leu Trp 260 265 270 Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr Ala Ala Leu 275 280 285 Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Leu Phe Cys Cys Arg Leu 290 295 300 Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn Ala Ser Gln 305 310 315 320 Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gln Gln Glu Glu Lys Pro 325 330 335 Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr 340 345 18 1191 DNA Homo sapiens CDS (1)...(1077) 18 atg gca ccc aat ggc aca gcc tct tcc ttt tgc ctg gac tct acc gca 48 Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala 1 5 10 15 tgc aag atc acc atc acc gtg gtc ctt gcg gtc ctc atc ctc atc acc 96 Cys Lys Ile Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile Thr 20 25 30 gtt gct ggc aat gtg gtc gtc tgt ctg gcc gtg ggc ttg aac cgc cgg 144 Val Ala Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg 35 40 45 ctc cgc aac ctg acc aat tgt ttc atc gtg tcc ttg gct atc act gac 192 Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp 50 55 60 ctg ctc ctc ggc ctc ctg gtg ctg ccc ttc tct gcc atc tac cag ctg 240 Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu 65 70 75 80 tcc tgc aag tgg agc ttt ggc aag gtc ttc tgc aat atc tac acc agc 288 Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser 85 90 95 ctg gat gtg atg ctc tgc aca gcc tcc att ctt aac ctc ttc atg atc 336 Leu Asp Val Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile 100 105 110 agc ctc gac cgg tac tgc gct gtc atg gac cca ctg cgg tac cct gtg 384 Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val 115 120 125 ctg gtc acc cca gtt cgg gtc gcc atc tct ctg gtc tta att tgg gtc 432 Leu Val Thr Pro Val Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val 130 135 140 atc tcc att acc ctg tcc ttt ctg tct atc cac ctg ggg tgg aac agc 480 Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser 145 150 155 160 agg aac gag acc agc aag ggc aat cat acc acc tct aag tgc aaa gtc 528 Arg Asn Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Lys Val 165 170 175 cag gtc aat gaa gtg tac ggg ctg gtg gat ggg ctg gtc acc ttc tac 576 Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr 180 185 190 ctc ccg cta ctg atc atg tgc atc acc tac tac cgc atc ttc aag gtc 624 Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Lys Val 195 200 205 gcc cgg gat cag gcc aag agg atc aat cac att agc tcc tgg aag gca 672 Ala Arg Asp Gln Ala Lys Arg Ile Asn His Ile Ser Ser Trp Lys Ala 210 215 220 gcc acc atc agg gag cac aaa gcc aca gtg aca ctg gcc gcc gtc atg 720 Ala Thr Ile Arg Glu His Lys Ala Thr Val Thr Leu Ala Ala Val Met 225 230 235 240 ggg gcc ttc atc atc tgc tgg ttt ccc tac ttc acc gcg ttt gtg tac 768 Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr 245 250 255 cgt ggg ctg aga ggg gat gat gcc atc aat gag gtg tta gaa gcc atc 816 Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Glu Val Leu Glu Ala Ile 260 265 270 gtt ctg tgg ctg ggc tat gcc aac tca gcc ctg aac ccc atc ctg tat 864 Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr 275 280 285 gct gcg ctg aac aga gac ttc cgc acc ggg tac caa cag ctc ttc tgc 912 Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Leu Phe Cys 290 295 300 tgc agg ctg gcc aac cgc aac tcc cac aaa act tct ctg agg tcc aac 960 Cys Arg Leu Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn 305 310 315 320 gcc tct cag ctg tcc agg acc caa agc cga gaa ccc agg caa cag gaa 1008 Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gln Gln Glu 325 330 335 gag aaa ccc ctg aag ctc cag gtg tgg agt ggg aca gaa gtc acg gcc 1056 Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr Ala 340 345 350 ccc cag gga gcc aca gac agg taaaagctcc aggtgtggag tgggacagaa 1107 Pro Gln Gly Ala Thr Asp Arg 355 gtcacggccc cccagggagc cacagacagg taagcgctga acagagactt ccgcaccggg 1167 taccaacagc tcttctgctg cagg 1191 19 359 PRT Homo sapiens 19 Met Ala Pro Asn Gly Thr Ala Ser Ser Phe Cys Leu Asp Ser Thr Ala 1 5 10 15 Cys Lys Ile Thr Ile Thr Val Val Leu Ala Val Leu Ile Leu Ile Thr 20 25 30 Val Ala Gly Asn Val Val Val Cys Leu Ala Val Gly Leu Asn Arg Arg 35 40 45 Leu Arg Asn Leu Thr Asn Cys Phe Ile Val Ser Leu Ala Ile Thr Asp 50 55 60 Leu Leu Leu Gly Leu Leu Val Leu Pro Phe Ser Ala Ile Tyr Gln Leu 65 70 75 80 Ser Cys Lys Trp Ser Phe Gly Lys Val Phe Cys Asn Ile Tyr Thr Ser 85 90 95 Leu Asp Val Met Leu Cys Thr Ala Ser Ile Leu Asn Leu Phe Met Ile 100 105 110 Ser Leu Asp Arg Tyr Cys Ala Val Met Asp Pro Leu Arg Tyr Pro Val 115 120 125 Leu Val Thr Pro Val Arg Val Ala Ile Ser Leu Val Leu Ile Trp Val 130 135 140 Ile Ser Ile Thr Leu Ser Phe Leu Ser Ile His Leu Gly Trp Asn Ser 145 150 155 160 Arg Asn Glu Thr Ser Lys Gly Asn His Thr Thr Ser Lys Cys Lys Val 165 170 175 Gln Val Asn Glu Val Tyr Gly Leu Val Asp Gly Leu Val Thr Phe Tyr 180 185 190 Leu Pro Leu Leu Ile Met Cys Ile Thr Tyr Tyr Arg Ile Phe Lys Val 195 200 205 Ala Arg Asp Gln Ala Lys Arg Ile Asn His Ile Ser Ser Trp Lys Ala 210 215 220 Ala Thr Ile Arg Glu His Lys Ala Thr Val Thr Leu Ala Ala Val Met 225 230 235 240 Gly Ala Phe Ile Ile Cys Trp Phe Pro Tyr Phe Thr Ala Phe Val Tyr 245 250 255 Arg Gly Leu Arg Gly Asp Asp Ala Ile Asn Glu Val Leu Glu Ala Ile 260 265 270 Val Leu Trp Leu Gly Tyr Ala Asn Ser Ala Leu Asn Pro Ile Leu Tyr 275 280 285 Ala Ala Leu Asn Arg Asp Phe Arg Thr Gly Tyr Gln Gln Leu Phe Cys 290 295 300 Cys Arg Leu Ala Asn Arg Asn Ser His Lys Thr Ser Leu Arg Ser Asn 305 310 315 320 Ala Ser Gln Leu Ser Arg Thr Gln Ser Arg Glu Pro Arg Gln Gln Glu 325 330 335 Glu Lys Pro Leu Lys Leu Gln Val Trp Ser Gly Thr Glu Val Thr Ala 340 345 350 Pro Gln Gly Ala Thr Asp Arg 355 

What is claimed is:
 1. An isolated nucleic acid consisting essentially of a nucleotide sequence selected from the group consisting of SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, and SEQ ID NO:15.
 2. A method of amplifying a nucleic acid, the method comprising hybridizing the isolated nucleic acid of claim 1 to a target nucleic acid, extending the 3′ end of the isolated nucleic acid and amplifying the extension product using a polymerase, whereby the target nucleic acid is a template for the extending.
 3. The method of claim 2, further comprising denaturing the extended isolated nucleic acid and repeating the hybridizing and extending steps.
 4. A method of sequencing a nucleic acid, the method comprising hybridizing the isolated nucleic acid of claim 1 to a target nucleic acid, extending the 3′ end of the isolated nucleic acid using a polymerase, in the presence of dideoxy nucleotides whereby the target nucleic acid is a template for the extending and determining the nucleotide sequence of the extension product.
 5. A method of detecting the presence of a nucleic acid encoding a human H2 receptor, the method comprising performing the method of claim 2 to form an amplified product, isolating nucleic acids in the amplified product on a gel by electrophoresis, and determining the presence of a band at a pre-determined position on the gel, wherein the presence of the band at the pre-determined position indicates the presence of the nucleic acid encoding the human H2 receptor.
 6. A kit comprising the nucleic acid of claim
 1. 7. The kit of claim 6, further comprising a restriction endonuclease.
 8. A method of detecting a polymorphism in a nucleic acid encoding a human H2 receptor, the method comprising hybridizing the nucleic acid of claim 1 to a target nucleic acid to form an at least partially doublestranded hybrid nucleic acid, isolating the hybrid nucleic acid on a gel by electrophoresis, and determining the presence of a band at a pre-determined position on a gel, wherein the presence of the band at the pre-determined position indicates the presence of the polymorphism.
 9. The method of claim 8, further comprising amplifying the target nucleic acid before the hybridizing step.
 10. A method of detecting a polymorphism in a nucleic acid encoding a human H2 receptor, the method comprising determining, by single-stranded conformational polymorphism analysis, whether the nucleic acid comprises SEQ ID NO:18 with at least one nucleotide substitution selected from the group consisting of T to C at position 398, A to T at position 525, A to G at position 620, A to G at position 649, A to G at position 692, and G to A at position 802, each nucleotide position being relative to SEQ ID NO:18. 